The overall goal of this research is to develop a molecular mechanistic understanding of the assembly, sorting and transport of the vacuolar-type proton-translocating ATPase (V-ATPase) in the simple model eukaryote, the yeast Saccharomyces cerevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analysis in yeast has identified a group of genes encoding proteins that function in the in the endoplasmic reticulum (ER) in assembly of the membrane sector of the V-ATPase. These V-ATPase assembly factors will be characterized by genetic and biochemical approaches, to investigate the interactions of the assembly factors with the V-ATPase membrane sector subunits in the ER. We have also identified possible ER-localized cargo receptors for loading the V-ATPase into COPII vesicles budding from the ER. The cargo receptors will be investigated for direct interactions with the V- ATPase subunits and assembly factors, and for their precise role in the process. Understanding the assembly of a complex, multisubunit integral membrane protein and its loading into vesicles exiting the ER is a fundamental issue in cell biology. There are two different forms of the yeast V-ATPase; the Golgi and endosomal form of the complex assembles with the Stv1p isoform of the 100 kDa subunit, and the complex on the vacuole membrane assembles with the Vph1p isoform of the 100 kDa subunit. We have identified mutations in the Stv1p N-terminal domain that lead to mislocalization of the Stv1-associated V-ATPase to the vacuole. We will characterize these mutations to determine whether they affect retention in the Golgi complex or retrieval of Stv1p back from the endosome. We have also identified a large group of genes involved in the sorting and retention of the Stv1- associated V-ATPase in the Golgi/endosome network, and we will characterize their encoded proteins to assess whether they bind to the Stv1p sorting/retention signals or generally affect the retention/retrieval of a larger group of Golgi membrane proteins. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of membrane transport and organelle biogenesis in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic studies in membrane trafficking and organelle acidification in yeast are providing important insights into our understanding of many diseases i humans related to defects in organelle acidification and protein mislocalization. PUBLIC HEALTH RELEVANCE: The overall goal of this research is to understand the role of the vacuolar-type ATPase (V-ATPase) in the acidification of cellular organelles in the simple model eukaryote, the yeast Saccharomyces cerevisiae. We will investigate how the V-ATPase, which is composed of 14 different protein subunits, is assembled in one compartment of the yeast cell and then transported with great fidelity to different cellular compartments. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of organelle acidification in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic studies in yeast are providing important insights into our understanding of many diseases in humans related to defects in organelle acidification. Understanding V-ATPase function will provide important insights into diseases of the kidney (renal tubular acidosis), bone diseases (osteopetrosis), and in tumor metastasis, and new drug targets should arise from our studies.